System in Salmonella typhimurium - Journal of Bacteriology

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described elsewhere (R. Menzel and J. Roth, manu- script in preparation), and strains TT1137, TT184, and. TT206 were obtained from John Roth (University of.
JOURNAL OF BACTERIOLOGY, Mar. 1980, p. 1071-1076 0021-9193/80/03-1071/06$02.00/0

Vol. 141, No. 3

Biochemistry and Regulation of a Second L-Prohne Transport System in Salmonella typhimurium RICHARD R. ANDERSON,' ROLF MENZEL,2 AND JANET M. WOOD' Guelph- Waterloo Centre for Graduate Work in Chemistry, University of Guelph, Guelph, Ontario, Canada NIG 2W1,1 and Biology Department, Howard Hughes Medical Research Institute, University of Utah, Salt Lake City, Utah 841122

This paper reports some biochemical characteristics of a second L-prohne transport system in Sabnonella typhimurium. In the accompanying paper, R.

Menzel and J. Roth (J. Bacteriol. 141:1064-1070, 1980) have identified this

system by showing that it is inactivated by mutations at the locusproP. We have found that it is an active transport system with an apparent Km for L-proline of 3 x 10' M and a strict specificity for L-proHne and some of its analogs. Unlike the L-prohne transport system encoded in putP, this second system is induced by

amino acid limitation.

Salmonella typhimurium possess two routes for the active accumulation of L-prohne. We designate as L-prohne permease I, or PP-I, the transport system that is eliminated by mutations at putP. The putP gene is part of a gene cluster that also includes putA, the gene encoding the polypeptide which possesses both L-proline oxidase and A'-pyrroline carboxylate dehydrogenase activities (12; R. Menzel, unpublished data). Expression of the put genes is subject both to catabolite repression and to specific induction by L-proline (11, 18). Menzel and Roth have used genetic techniques to identify a second L-proline transport system, PP-II, that is eliminated by mutations at proP (7). We have used strains of S. typhimurium defective in PP-I to examine the kinetics, specificity, and energy dependence of PP-II. Measurements of L-proline uptake activity as a function of bacterial growth conditions suggest that induction of PP-II is part of the stringent response to amino acid starvation.

of the other strains is described by Menzel and Roth (7). Materials. 3,4-Dehydro-DL-proline and 4,5-dehydro-L-pipecolate (L-baikiain) were purchased from Calbiochem (San Diego, Calif.). Nutritional supplements, antibiotics, L-azetidine-2-carboxylic acid, 4-hydroxy-L-proline, L-thiazolidine-4-carboxylic acid, and D-melibiose were from Sigma Chemical Co. (St. Louis, Mo.). Microbiological media were from Difco Laboratories (Detroit, Mich.), and L-[U-_4C]glutamine and L-[U-'4C]proline were from New England Nuclear Corp. (Boston, Mass.). All other reagents were of the highest grade available. Cultivation of bacteria. Liquid cultures were prepared by using MOPS minimal salts medium (8) with D-fructose (2 mg/ml) as carbon source and NH4Cl (0.54 mg/ml) as nitrogen source. MOPS medium contains the following major constituents: KH2PO4, 1.32 mM; MgCl2, 0.523 mM; K2SO4,0.276 mM; FeSO4, 0.01 mM; NaCl, 50 mM; morpholinopropane sulfonate, 40 mM; and Tricine, 4 mM. Its pH is 7.2. Bacteria grown for transport measurements were always subcultured and harvested during logarithmic growth as previously described (17), although not all cultures reached an optical density of 1.0 (see Results). Culture mass doubling times were determined by monitoring the abMATERIALS AND METHEODS sorbance at 600 nm (A600) of triplicate 2.5-ml cultures Bacterial strains. The strains used in this study, in standardized test tubes (13 by 100 mm) which were all derivatives of S. typhimurium LT2 or LT7, are shaken at 200 rpm. All cultures were grown at 37°C. listed in Table 1. Strains TT3329, TT3330, and TT3331 Uptake assays. Initial rates of aerobic amino acid were constructed by transducing strain TT2612 to uptake were measured as described by Berger and tetracycline resistance with phage P22 grown on the Heppel (2) with the following modifications: all preinauxotrophic donors TT1137, TT184, and TT206, re- cubations were performed at room temperature, and spectively. The isolation of strain TT2612 will be MOPS minimal medium (8) replaced medium A. The described elsewhere (R. Menzel and J. Roth, manu- transport energy source was D-glucose (2 mg/ml), and script in preparation), and strains TT1137, TT184, and the substrate concentrations were 10 uM for L-glutaTT206 were obtained from John Roth (University of mine and 200,uM for L-proline unless otherwise indiUtah, Salt Lake City). Transductants were selected on cated. Uptake of radioactivity was always a linear nutrient agar containing tetracycline at 25 ,ig/ml, and function of assay time period. All uptake experiments the auxotrophic requirements of strains TT3329, were performed in duplicate or triplicate, and all valTT3330, and TT3331 were confirmed by nutritional ues quoted represent averages of results from two or tests on minimal media. The isolation and construction more such experiments unless otherwise indicated. 1071

1072

TABLE 1. Bacterial strainse Strain

desiga-

J. BACTERIOL.

ANDERSON, MENZEL, AND WOOD

Genotype

tion

Derovativeof wild

type LT7 LT2 LT2 LT2 LT2 LT2 LT2

proAB47 putPA523 proAB47putPA523 proAB662::TnlO leull5I::TnlO hisH8677::TnlO proAB47putPA523 proP673 zjd-27::TnlO LT2 TT2612 putP853::Tn5 LT2 TT3329 putP853.:Tn5 hisH8677::TnlO LT2 TT3330 putP853::Tn5proAB6621.:TnlO TT3331 putP853::Tn5 leull15::TnlO LT2 aGenetic nomenclature is that defined by Sanderson and Hartman (14). proAB47 TR4910 TR5281 TT184 TT206 TT1137 TT1801

The error bars in Fig. 1 and 2 represent standard deviations of the indicated mean value. Analysis of intracellular radioactivity. To assess the fate of accumulated radioactive L-prohne, the small-molecule pool of the bacteria was extracted and analyzed by thin-layer chromatography and autoradiography as previously described (18). Protein assay. The protein content of cell suspensions was measured by the method of Lowry et al. (6), using bovine serum albumin as a standard. Analysis of kinetic data. Kinetic data were analyzed by nonlinear least-squares regression, using program BMDO7R of the BMD biomedical computer programs (3). The relative quality of fit to the data by equations (1) to (3) (see Results) was assessed by applying the F test for goodness of fit and by considering the significance of the parameter values. Asymptotic standard deviations are provided as error limits on all parameter values cited in the text.

RESULTS

Regulation of L-proline uptake via PP-II. Menzel and Roth have shown that, when Lproline uptake activity is measured with 2 x 10' M substrate, PP-Il contributes only 5 to 10% of the total uptake activity of S. typhimu-

doubling times for cultures of strains TR5281 and TT3330 increased from 60 min to 284 and 197 min, respectively, when the medium L-proline concentration was 2 ,zg/ml rather than 2 mg/ml. This very dramatic change in growth rate may result from the absence of PP-I in these strains. It was accompanied by a threefold increase in L-proline uptake activity (uptake measured at 2 x 10-4 M L-proline for reasons described below). The uptake rates achieved in this way were comparable with rates of uptake via PP-I in prototrophic bacteria grown on Dfructose or D-glucose plus L-proline (18). L-Prohne uptake was also measu.red in the Lhistidine auxotroph TT3329 (putP853::Tn5 hisH8677::TnlO) and in the L-le icine auxotroph TT3331 (putP853::Tn5 leull51::TnlO) grown under conditions of amino acid exces$ and amino acid starvation (Table 2). Growth in medium containing very low concentrations of L-histidine or L-leucine did not alter the mass doubling time for cultures of these strains, though it did reduce the extent of growth for strain TT3331 (final A6w = 0.3). Harvesting these cultures at the late log phase of growth presumably yielded bacteria more strongly limited for their auxotrophic requirements than is indicated by their doubling times. Again, amino acid starvation produced a two- to threefold enhancement in L-prohne uptake activity in comparison with unstarved controls (Table 2). The induction of PP-II is therefore part of a general response to amino acid starvation rather than a specific response to Lproline limitation. Kinetics of L-proline uptake via PP-II. Figure 1 illustrates the variation in L-proline uptake activity of strains TT1801, TR4910, and TABLE 2. Induction of L -proline uptake via PP-II S.

typhi-

murium strain

Auxotrophic Growth mea dium requirement

Mass dou-

blmng

time"

L-Proline uptakec

(min) 4.2 63 TR5281 L-Proline MOPS-HP 284 13.7 MOPS-LP 4.3 MOPS-HP 61 TT3330 L-Prohne 197 11.4 MOPS-LP 64 4.8 TT3329 L-Histidine MOPS-HH MOPS-LH 65 10.6 67 5.2 TT3331 L-Leucine MOPS-HL 72 MOPS-LL 14.9 a MOPS-HP, MOPS-HH, and MOPS-HL contained 2 mg of L-proline, L-histidine, and L-leucine per ml, respectively; MOPS-LP, MOPS-LH, and MOPS-LL contained 2 pug of Lproline. 4 ,ug of L-histidine, and 4 pg of L-leucine per ml, respectively, in addition to D-fructose and NH4+. b Estimated by monitoring culture A6e as described in the

rium grown on miniimal medium containing Dglucose and L-proline (7). To study in isolation the dependence of PP-II activity on bacterial growth conditions, we used strains of S. typhimurium lacking PP-I. Prototrophic putP strains showed no significant variation in L-proline uptake activity if succinate replaced D-fructose or D-glucose as sole carbon source. Uptake activity also remained unaltered if bacteria were grown in L-proline-supplemented media containing either carbon source (data not shown). However, strains auxotrophic for L-proline and defective in PP-I did show a striking increase in L-proline text. 'Initial rates of L-proline uptake, measured as described in uptake activity when their growth was limited the text, are given as nanomoles minute-' (milligram of proby the supply of L-proline (Table 2). The mass tein)'.

SECOND L-PROLINE TRANSPORT SYSTEM

VOL. 141, 1980

I1: F

I 0

0.2

0.4 0.6 L-PROLINE CONCENTRATION (mlil)

0.0

1.0

FIG. 1. Kinetics of L -proline uptake. Initial rates of L -proline uptake were measured as a function of L -proline concentration for strains TR5281 (U), TT1801 (0), and TR4910 (A) (see text for growth conditions). The data were derived from experiments using at least two separate ceU preparations in each case, and the lines drawn are those derived from regression analysis of these data (see text). The inset shows a plot of s/v versus s for the data from strains TR4910 and TR5281 after subtraction of the background uptake component (vi) that was estinated by using strain 171801. The lines are derived from the saturable component of the regression equation.

TR5281 for a series of L-proline concentrations from 5 x 10-7 to 1 X 10-3 M. Strain TT1801, which lacks both PP-I and PP-II, was supplied with glycyl-L-proline (2 mg/ml) in order to meet its L-proline requirement via a dipeptide uptake system. In spite of its genetic defects, strain TT1801 could perform the time-dependent uptake of radioactive L-proline. This residual uptake activity may be due to an additional uptake system, or it may represent residual PP-II uptake activity (proP673 contains a point mutation that is subject to spontaneous reversion). The initial rate of L-proline uptake by this strain showed a linear dependence on L-prohne concentration in the range studied, however, so the data were fit to the relationship: (1) vb=Ks where Vb is background uptake rate in nanomoles minute-' (milligram of protein)-', s is L-proline concentration (molar), and K is found to be 4.9 x 103 ± 0.2 x 103 nmol min-' (mg of protein)-' M-'. This value for Khas been used to represent background uptake activity in analyzing the kinetics of proline uptake by strains TR4910 and TR5281. The uptake activity of strain TR4910 was examined to provide a control that was uninduced but had an intact PP-II system. Although

1073

it lacks PP-I, it is prototrophic and hence unlikely to contain a reduced cytoplasmic pool of L-proline or any other amino acid. The data indicated some saturation of uptake activity with increasing L-proline concentration, though true saturation was not reached within this concentration range (Fig. 1). The data were fit to equation (2): V = [VVmS/(Km + s)] + (4.9 x 103)s (2) which takes into account the background uptake component illustrated by strain TT1801. In this equation, v is initial uptake rate in nanomoles minute-' (milligram of protein)-', s is as defined above, and the parameter values Vm = 10 ± 1 nmol min-' (mg of protein)'- and Km = 4 x 10-4 ± 1 X 1o-4 M yield the regression lines shown in Fig. 1. These data fit equation (2) significantly better than either equation (1) or the simple Michaelis-Menten relationship: v = Vms/(Km + s) (3) Finally, the kinetics of the induced PP-II system have been measured by using the auxotrophic strain TR5281 grown under conditions of amino acid limitation (2 ,ug of L-proline per ml supplied). L-Proline uptake by this strain was also a partially saturable function of L-proline concentration. Fitting these data to equation (2) yielded the parameter values Vm = 32 ± 1 nmol min-' (mg of protein)-' and Km = 3.4 x 10-4 ± 0.2 x 1O-4 M, which defmed the regression lines shown in Fig. 1 and again produced a significant improvement in fit over equation (3). This estimate for Km agrees with that derived from strain TR4910 above, and a comparison of the estimates of Vm again illustrates the threefold induction in uptake activity achieved by amino acid starvation. Fate of L-proline accumulated via PP-II. To assess the fate of L-proline accumulated via PP-II, the intracellular radioactive compounds present after a standard uptake period were extracted and analyzed (Table 3). In strain proAB47, which possess intact put genes, much of the radioactive L-prohne accumulated during a 15-s uptake period was catabolized to Al-pyrroline carboxylate and L-glutamate. In strain TR5281, the L-proline taken up via PP-II remained unaltered since the catabolic enzymes encoded byputA are absent, as is PP-I. By using the uptake values for strain TR5281, we could calculate approxiimate intracellular L-proline concentrations (Table 3). These calculations show that cells induced for PP-II could accumulate L-proline to a concentration approximately 100-fold greater than that of their medium after 15 s of uptake, a clear indication that transport via PP-II is active.

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ANDERSON, MENZEL, AND WOOD

TABLE 3. Fate of L -proline accumulated via PP-IH S. tshiram strain

Growthmediuma

Uptake rateb

Extraction efficiencyc P CPM post Efficiency CPM pre

proline concnd

(M)

88 N.C. 307 2613 4.8 MOPS-HP proAB47 97 N.C. 449 17222 40.0 MOPS-LP proAB47 73 0.001 385 1435 2.4 MOPS-HP TR5281 382 99 0.025 29080 74.4 MOPS-LP TR5281 a, b See Table 2. ' for 15 s under the conditions of the standard Cells were permitted to take up L-proline (200,tM; 25 uptake assay. CPM pre and CPM post are the accumulated radioactive counts before and after extraction of the intracellular small-molecule pool (see text). d The cytoplasMic L-proline concentration after 15 s of uptake was calculated assuming a cytoplasmic water content of 0.73 id1 per mg of protein (16). Intracellular L-proline concentrations were not calculated for strain proAB47 because of L-prohne catabolism (see text).

pCi/pmol)

Effects of metabolic inhibitors. L-Proline uptake via PP-II (like L-glutamine uptake) was reduced if D-glucose was omitted from the uptake assay mixture (Table 4). Both an uncoupler of oxidative phosphorylation (2,4-dinitrophenol) and an inhibitor of ATP synthesis (arsenate) markedly inhibited D-glucose supported uptake via PP-II, though the former was the more effective inhibitor. Predictably, L-glutamine uptake was strongly inhibited by arsenate and slightly reduced by 2,4-dinitrophenol. These results provide further evidence that uptake via PP-II is active. Specificity of uptake via PP-Il. The specificity of PP-II was assessed by testing the ability of a variety of molecules to inhibit L-[14C]proline uptake via this system. Table 5 summarizes the effects on L-proline uptake of the commonly occurring amino acids and of D-melibiose and L-thiazolidine-4-carboxylate, each present in the assay mixture at a concentration 20-fold greater than that of the substrate. None of these compounds strongly inhibited L-proline uptake. Four other L-proline analogs did inhibit L-proline uptake more strongly, and their effects were tested at a variety of inhibitor concentrations (Fig. 2). L-Azetidine-2-carboxylate and 3,4-dehydro-DLproline were both strong inhibitors of L-proline uptake, reducing uptake activity to one-half of its control level when present at approximately 0.3 and 0.7 mM, respectively. 4,5-Dehydro-L-pipecolate (L-baikiain) and 4-hydroxy-L-proline also inhibited uptake, albeit much more weakly. Only about 40% inhibition of uptake was achieved with each of these compounds even when they were present in 20-fold excess (4 mM). System PP-II is thus specific for L-proline and a limited number of its close structural analogs.

TABLE 4. Effects of metabolic inhibitors on L -proline and L -glutamine uptake uptake L-Prohne uptake

Additions to uptake

L-Glutamine take upof

nmol

%of

mg'

control

None Glucose Glucose + DNP

2.8 16.6 4.6

17 100 28

1.4 3.8 3.1

37 100 82

None Glucose Glucose +

3.6 18.2 8.3

20 100 46

1.3 3.6 0.7

36 100 19

assaya

mol

control

A

B

arsenate

Uptake assays were performed as described in the text, using strain TR5281 grown in MOPS-LP (defined in Table 1). The data cited represent means of triplicate assays from a single experiment that was replicated. (A) Assays were performed with the addition of no carbon source, D-glucose (11 mM), or D-glucose plus 2,4-dinitrophenol (DNP, 0.5 mM) as indicated. (B) Uptake assays were performed in medium lacking phosphate and using cells washed with phosphate-free MOPS medium. No carbon source, D-glucose (11 mM), or n-glucose plus sodium arsenate (0.5 mM) were added as indicated. a

mutations in putP, S. typhimurium possesses a second L-proline uptake activity, PP-I1, associated with the locus proP. PP-II has a relatively high uptake Km (3 x 1i-' M; Fig. 1) compared with that of PP-I (2 x 10'- M as measured in Escherichia coli [18]) or with those of other amino acid transport systems. The specificities of the two systems as determined by uptake inhibition studies are similar. Neither system interacts with any of the 19 commonly occurring a-amino acids, and both are strongly inhibited by the L-proline analogs L-azetidine-2-carboxylDISCUSSION ate and 3,4-dehydro-DL-proline (Table 5 and Fig. In addition to the recognized L-proline trans- 2). Unlike PP-I, however, PP-Il is not inhibited port system, PP-I, which may be inactivated by by L-thiazolidine-2-carboxylate and it is in-

VOL. 141, 1980

SECOND L-PROLINE TRANSPORT SYSTEM

hibited, though weakly, by 4-hydroxy-L-proline and 4,5-dehydro-L-pipecolate (10, 18). Like PPI, PP-II is an active uptake system; it is capable of producing an intracellular L-proline concentration 100-fold greater than that of its medium (Table 3), and its activity is stimulated in the presence of D-glucose (Table 4). L-Prohne uptake via PP-II is inhibited by 2,4-dinitrophenol and, less strongly, by arsenate. Further studies will be required, however, to determine whether PP-II shows the structural and energetic features characteristic of osmotic shock-sensitive or osmotic shock-resistant transport systems. No proline uptake activity with the kinetic characteristics of PP-Il has been identified during transport studies using cytoplasmic membrane vesicles derived from E. coli (4, 5). However, the bacteria from which the vesicles were prepared were not grown in a manner that would induce PP-II. The most novel characteristic of PP-II is its regulation. A significant increase in PP-II activity is observed in amino acid-starved bacteria (Table 2 and Fig. 1). Attempts to further amplify this activity by growing strain TR5281 in continuous culture failed to yield stable bacterial populations under conditions of L-proline limitation. However, L-prohne uptake activities as much as 30-fold greater than those of the unstarved bacteria were observed [90 nmol min-' (mg of protein)-']. Quay and Oxender have observed an induction of the branched-chain amino acid uptake systems, LIV-I and LIV-II, that is caused by amino acid starvation and requires the relA gene product (9, 10). We are currently examining the dependence of PP-II induction on reA. Our data and those of Quay and Oxender suggest that, as predicted by Stephens et al. (15), amino acid transport is induced along with some amino TABLE 5. Specificity of uptake via PP-II Relative uptake

Inhibitor

Inhibitor

(%)a

(%)a

Nil L-Alanine

100 ....

L-Arglinie L-Asparagine

L-Aspartate L-Cysteine L-Glutamate L-Glutamine Glycine ...... L-Histidine ... L-Isoleucine

83 92 79 86 78 91 90 97 110 104

Relative Uptake

L-Leucine L-Lysine

105 .......

L-Methione

L-Phenylalanine L-Serine L-Threonine

L-Tryptophan L-Tyrosine ..... L-Valine ....... L-Melibiose ....

L-Thiazolidine-4carboxylate

84 96 92 81 90 102 93 89 92 72

a L-Proline uptake was measured as described in the text, using 200 /AM L-proline as substrate. Inhibitors were present at 4 mM. The uninhibited uptake rate was 8.0 nmol min-' (mg of protein)-'.

laDO

1075

L-azetidine-2-carboxylate

-

3,4-dehydro-D,L-proline

-

a

10

D0

so O

atS%

DO -

4,5-dohydro- L-pipecolate

9

10

1I DO

4-hydroxy-L-prollne

so~~

-

_

WV

0

1

2

3

4

Inhibitor Concentration (mM)

FIG. 2. Inhibitors of L -proline uptake. Initial rates of L -proline uptake were measured as described in the text, adding the indicated L -proline analogs to the cells simultaneously with the radioactively labeled substrate. Uptake activity is given as a percentage of the uninhibited control value, 16 nmol min-1 (mg ofprotein) 1.

acid biosynthetic enzymes as part of the stringent response to amino acid starvation. Genetic complementation studies suggest that the put and pro genes are common to S. typhimurium and E. coli (7, 12), and it seems likely that transport systems PP-I and PP-II are present in a wider range of enterobacteria. It is tempting to suggest that the two transport systems play complementary physiological roles in these organisms, PP-I supplying L-prollne primarily for oxidation by L-prohne oxidase/Al-pyrroline carboxylate dehydrogenase and PP-II providing L-proline for protein synthesis. (An analogous suggestion has been made with respect to histidine transport [1].) This hypothesis leads to the prediction that PP-I will have a low affinity for proline since it will function when proline is abundant, whereas PP-Il will be a high-affinity system designed to scavenge proline, even at low concentrations, from the growth medium. Unfortunately, these predictions are

ANDERSON, MENZEL, AND WOOD 1076 not filfilled by our observations of uptake Km for the two systems; the Km for proline uptake by PP-II is 100-fold higher than that for uptake via PP-I. The expression and interdependence of the two uptake systems when both are present in a genetically intact organism has yet to be examined. It is possible that PP-I activity is reduced below the significant basal levels that are normally observed in L-proline prototrophic strains if the cytoplasmic concentration of the inducer, L-proline, is reduced during specific or general amino acid starvation. Under such conditions, uptake via PP-Il, in spite of its high K,,, would assume a new importance. It is clear that both PP-I and PP-Il must now be considered in evaluating the response of L-proline uptake activity to variations in nutrient supply. ACKNOWLEDGMENTS We are grateful to the National Research Council of Canada and to Research Corporation for their financial support. R.R.A. is a research fellow of the National Research Council of Canada. Thanks are also due to Paul Galatsis and Maureen Clancy for their technical assistance, to John Roth for discussions of the data, and to Flo Rayner and Sarah Edwards for their assistance in preparation of the manuscript.

LITERATURE CITED 1. Ames, G. F-L. 1972. Components of histidine transport, p. 409-426. In C. F. Fox (ed.), Membrane research. Academic Press Inc., New York. 2. Berger, E. A., and L A. Heppel. 1974. Different mechanisms of energy coupling for the shock sensitive and shock resistant amino acid permeases of Escherichia coli. J. Biol. Chem. 249:7747-7755. 3. Dixon, W. J. (ed.). 1976. BMD biomedical computer programs. University of California Press, Berkeley and Los Angeles. 4. Kaback, H. R., and R. R. Stadtman. 1966. Proline uptake by an isolated cytoplasmic membrane preparation of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A.

J. BACTERIOL. 55:920. 5. Lombardi, F. J., and H. R. Kaback. 1972. Mechanisms of active transport in isolated bacterial membrane vesicles. VIII. The transport of amino acids by membranes prepared from Escherichia coli. J. Biol. Chem. 247: 7844-7857. 6. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 7. Menzel, R., and J. Roth. 1980. Identification and mapping of a second proline permease in SalnoneUa t)phimurium. J. Bacteriol. 141:1064-1070. 8. Neidhardt, F. C., P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J. Bacteriol. 119: 736-747. 9. Quay, S. C., and D. L Oxender. 1976. Regulation of branched-chain amino acid transport in Escherichia coli. J. Bacteriol. 127:1225-1238. 10. Quay, S. C., and D. L Oxender. 1979. The reU locus specifies a positive effector in branched-chain amino acid transport regulation. J. Bacteriol. 137:1059-1062. 11. Ratzkin, B., M. Grabnar, and J. Roth. 1978. Regulation of the major proline permease gene of Sabnonella typhimurium. J. Bacteriol. 133:737-743. 12. Ratzkin, B., and J. Roth. 1978. Cluster of genes controlling proline degradation in Salmonella typhinurium. J. Bacteriol. 133:744-754. 13. Rowland, I., and N. Tristram. 1975. Specificity of the proline transport system. J. Bacteriol. 123:871-877. 14. Sanderson, K. E., and P. E. Hartmanz 1978. Linkage map of Sabnonella typhimurium, edition V. Microbiol. Rev. 42:471-519. 15. Stephens, J. C., S. W. Artz, and B. N. Ames. 1975. Guanosine 5'-diphosphate-3'-diphosphate (ppGpp): positive effector for histidine operon transcription and general signal for amino acid deficiency. Proc. Natl. Acad. Sci. U.S.A. 72:4389-4393. 16. Winkler, H. H., and T. H. Wilson. 1966. The role of energy coupling in the transport of f-galactosides by Escherichia coli. J. Biol. Chem. 241:2200-2211. 17. Wood, J. M. 1975. Leucine transport in Escherichia coli: the resolution of multiple transport systems and their coupling to metabolic energy. J. Biol. Chem. 250:44774485. 18. Wood, J. M., and D. Zadworny. 1979. Characterization of an inducible porter required for L-proline catabolism by Escherichia coli K12. Can. J. Biochem. 57:1191.